A two-in-one electrochemical test paper for simultaneously detecting inorganic phosphorus and salicylic acid and a preparation method thereof

Abstract

The application discloses a two-in-one electrochemical test paper for simultaneously detecting inorganic phosphorus and salicylic acid and a preparation method thereof, and relates to the technical field of in vitro diagnosis electrochemical test paper. The electrochemical test paper comprises an insulating substrate, a conductive film layer, a reaction film layer, a liquid guide groove body and a hydrophilic film layer; the conductive film layer comprises a working electrode and a reference electrode, the working electrode comprises a first reactant working electrode for detecting inorganic phosphorus and a second reactant working electrode for detecting salicylic acid, the first reactant working electrode is provided with a first layer of reaction film, and the second reactant working electrode is provided with a second layer of reaction film. According to the application, two independent working electrodes are integrated on the same insulating substrate, the reaction film loaded with corresponding enzymes is modified on the two working electrodes respectively, the quantitative detection of two indexes of inorganic phosphorus and salicylic acid can be simultaneously realized on the same test paper, the sample consumption is reduced, the detection cost is reduced, and the application is suitable for clinical detection and popularization and application.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic electrochemical test paper technology, and in particular to a two-in-one electrochemical test paper for the simultaneous detection of inorganic phosphorus and salicylic acid and its preparation method. Background Technology

[0002] Inorganic phosphorus is an essential mineral for the human body, participating in key physiological processes: bone and teeth mineralization, energy metabolism, cell signaling, acid-base balance, and nucleic acid and phospholipid synthesis. The normal range for adult serum phosphorus is 0.81–1.45 mmol / L. Serum phosphorus <0.81 mmol / L is considered hypophosphatemia. Patients with chronic hypophosphatemia often experience loss of appetite, anorexia, nausea, and vomiting; severe cases may present with decreased gastric tone, paralytic ileus, and dysphagia. Severe hypophosphatemia, due to impaired energy metabolism, can lead to severe cardiomyopathy, decreased cardiac output, hypotension, and even congestive heart failure. Serum phosphorus >1.45 mmol / L is considered hyperphosphatemia. Hyperphosphatemia is a core complication of chronic kidney disease and requires regular monitoring.

[0003] Salicylic acid is an organic acid primarily used as an important raw material in fine chemicals such as pharmaceuticals, fragrances, dyes, pesticides, and rubber additives. It is also a precursor to aspirin (acetylsalicylic acid) and a plant hormone. The detection of salicylic acid in serum has clear toxicological significance in clinical practice, mainly used for the diagnosis and monitoring of salicylic acid-based drug (especially aspirin) poisoning, and in specific circumstances for monitoring therapeutic drugs.

[0004] Currently, elderly people often suffer from chronic kidney disease and antibiotic overdose, which can be diagnosed by testing two commonly used indicators: inorganic phosphorus and salicylic acid. The methods used are primarily wet-method testing, employing reagent kits and large biochemical analyzers, which is time-consuming, complex, and requires professional operation, making it inconvenient for users or patients. Dry-method testing of both indicators simultaneously lacks relevant product registration certificates and mature products on the market, thus the market is essentially untapped. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a two-in-one electrochemical test strip for the simultaneous detection of inorganic phosphorus and salicylic acid, and its preparation method, thus solving the problem that there is currently no electrochemical test strip capable of simultaneously detecting inorganic phosphorus and salicylic acid. The specific technical solution is as follows: A two-in-one electrochemical test strip for the simultaneous detection of inorganic phosphorus and salicylic acid, comprising: An insulating substrate includes a first insulating substrate and a second insulating substrate disposed opposite to each other; A conductive film layer, the conductive film layer comprising a first conductive film layer disposed on the inner surface of the first insulating substrate and a second conductive film layer disposed on the inner surface of the second insulating substrate; The first conductive film layer includes a first reactant working electrode and a second reactant working electrode that are insulated from each other. The second conductive film layer includes a first reactant reference electrode, a second reactant reference electrode, a first pin, and a second pin, which are mutually insulated from each other; The second insulating substrate has a first through hole and a second through hole. The first reactive working electrode is electrically connected to the first pin through the first through hole, and the second reactive working electrode is electrically connected to the second pin through the second through hole. The reaction membrane layer includes a first reaction membrane and a second reaction membrane, wherein the first reaction membrane covers the first reactant working electrode and the second reaction membrane covers the second reactant working electrode; and a reaction enzyme layer is disposed on the first reaction membrane and the second reaction membrane. A liquid guiding tank is disposed between the first insulating substrate and the second insulating substrate. The liquid guiding tank includes a first liquid guiding tank and a second liquid guiding tank that are independent of each other. The first liquid guiding tank is provided with a first sample inlet, and the second liquid guiding tank is provided with a second sample inlet. The first reaction membrane is located in the first liquid guiding tank, and the second reaction membrane is located in the second liquid guiding tank. A hydrophilic membrane layer, comprising a first hydrophilic membrane and a second hydrophilic membrane, which respectively cover the first liquid guiding groove and the second liquid guiding groove, for promoting capillary flow of the sample in the first liquid guiding groove and the second liquid guiding groove; The second insulating substrate is also provided with a first sample inlet observation window and a second sample inlet observation window. The first sample inlet observation window corresponds to the position of the first sample inlet, and the second sample inlet observation window corresponds to the position of the second sample inlet.

[0006] Further, the reaction enzyme layer of the first reaction membrane comprises: 25-35 KU / L hypoxanthine oxidase, 60-80 KU / L purine nucleoside phosphorylase, 1-10 g / L N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt, 1-10 g / L hypoxanthine nucleoside, 1-10 g / L film-forming agent, 1-20 g / L stabilizer, 1-10 g / L surfactant, 1-10 g / L electronic medium, and 0.1-0.5 M buffer solution.

[0007] Furthermore, the reaction enzyme layer of the second reaction membrane comprises: 15-20 KU / L salicylate hydroxylase, 50-100 KU / L tyrosinase, 1-10 g / L 3-methyl-2-benzothiazolinone hydrazone hydrochloride, 1-10 g / L reduced nicotinamide adenine dinucleotide, 1-10 g / L film-forming agent, 1-20 g / L stabilizer, 1-10 g / L surfactant, 1-10 g / L electronic medium, and 0.1-0.5 M buffer.

[0008] Furthermore, the film-forming agent is sodium alginate; the stabilizer is trehalose; and the surfactant is Triton X-100.

[0009] Furthermore, the electronic medium is potassium ferrocyanide; the buffer solution is phosphate buffer.

[0010] Furthermore, the conduction paths of the first reactant working electrode and the first reactant reference electrode are misaligned with the conduction paths of the second reactant working electrode and the second reactant reference electrode.

[0011] The present invention also provides a method for preparing the electrochemical test paper, comprising the following steps: (1) Mix and stir the raw materials of the enzyme layer of the first reaction membrane for 1 hour to obtain the first enzyme slurry; mix and stir the raw materials of the enzyme layer of the second reaction membrane for 1 hour to obtain the second enzyme slurry; fix the first enzyme slurry on the first reaction membrane and fix the second enzyme slurry on the second reaction membrane using an enzyme dispensing machine; (2) Print a first conductive film layer on a first insulating substrate, print a second conductive film layer on a second insulating substrate, and dry; (3) Fix the first reaction membrane containing enzyme slurry on the first conductive film layer; fix the second reaction membrane containing enzyme slurry on the second conductive film layer; attach the liquid guiding groove to the insulating substrate, and attach the hydrophilic film layer on the liquid guiding groove body to obtain the finished electrochemical test paper.

[0012] Furthermore, the drying temperature is 50-60℃, and the drying time is 20-30 minutes.

[0013] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention improves the structure and reactive enzyme layer of the electrochemical test strip, enabling it to be used with a multi-parameter analyzer for simultaneous, rapid, and quantitative detection of inorganic phosphorus and salicylic acid content in whole blood, serum / plasma, and other body fluids in humans or animals. This eliminates the need for separate preparation of two test strips, effectively saving detection time, reducing sample volume, and lowering detection costs. Furthermore, the detection results show good matching with biochemical analyzer results, demonstrating high efficiency. Testing shows that the R-squared value of the electrochemical test strip of this invention is above 0.99 compared to hospital biochemical values, indicating high result matching and suitability for widespread clinical application.

[0014] 2. In this invention, the first and second liquid guiding channels of the electrochemical test paper are independent of each other, each with its own independent sample inlet (first sample inlet and second sample inlet), and the first and second reaction membranes are isolated from each other to prevent interference. This allows the same test paper to simultaneously detect both inorganic phosphorus and salicylic acid. Furthermore, the working electrode (first conductive film layer) of the electrochemical test paper is disposed on the inner surface of the first insulating substrate; the reference electrode and leads (second conductive film layer) are disposed on the inner surface of the second insulating substrate. This face-to-face arrangement of the working electrode and reference electrode shortens the electrode spacing and improves the response speed and signal intensity of the electrochemical reaction.

[0015] 3. The reaction enzyme layer of the first reaction membrane of the present invention includes hypoxanthine oxidase, purine nucleoside phosphorylase, sodium N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, hypoxanthine nucleoside, etc., wherein inorganic phosphorus in the sample reacts with hypoxanthine nucleoside to generate hypoxanthine under the catalysis of purine nucleoside phosphorylase; hypoxanthine undergoes an oxidation reaction under the action of hypoxanthine oxidase, and the electrons released by the reaction are transferred to the working electrode through the electronic medium, generating a current signal proportional to the concentration of inorganic phosphorus. The analyzer calculates the content of inorganic phosphorus in the sample by converting the current intensity.

[0016] 4. The enzyme layer of the second reaction membrane of this invention includes salicylate hydroxylase, tyrosinase, 3-methyl-2-benzothiazolinone hydrazone hydrochloride, and reduced nicotinamide adenine dinucleotide, etc. Salicylic acid in the sample undergoes a specific oxidation reaction under the synergistic catalysis of salicylate hydroxylase and tyrosinase. The reaction process is accompanied by electron transfer, with electrons transferred to the electrode through the medium. The resulting current intensity is positively correlated with the salicylic acid concentration, and the instrument quantitatively calculates the salicylic acid concentration accordingly. Reduced nicotinamide adenine dinucleotide ensures that the enzyme-catalyzed reaction is at its maximum rate, improving substrate conversion efficiency, amplifying the current signal, enhancing the sensitivity of low-concentration detection, and also offsetting oxidative degradation losses during storage, maintaining the stability of the reaction system, and reducing numerical drift after long-term storage.

[0017] 5. The electrochemical test strip of this invention is simple to use, requires no professional operation, and provides fast, accurate, and reliable testing results. It is suitable for various pharmacies, clinics, research institutes, community hospitals, veterinary hospitals, testing centers, or other government-sponsored surveys and inspections, and is suitable for widespread application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is an exploded view of the test strip structure in Embodiment 1 of the present invention; Figure 2 This is a cross-sectional view of the test strip in Embodiment 1 of the present invention; Figure label: 1. First insulating substrate; 2. First conductive film layer; 2-1. First reactant working electrode; 2-2. Second reactant working electrode; 3. Reaction film layer; 3-1. First reaction film; 3-2. Second reaction film; 4. Liquid guiding tank; 4-1. First liquid guiding tank; 4-2. First sample inlet; 4-3. Second sample inlet; 4-4. Second liquid guiding tank; 5. Hydrophilic film layer; 5-1. First hydrophilic film; 5-2. Second hydrophilic film; 6. Second conductive film layer; 6-1. First sample inlet observation window; 6-2. Second sample inlet observation window; 6-3. First reactant reference electrode; 6-4. Second reactant reference electrode; 6-5. First pin; 6-6. Second pin; 7. Second insulating substrate; 7-1. First through hole; 7-2. Second through hole. Detailed Implementation

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0021] Example 1 like Figure 1 and Figure 2 As shown, a two-in-one electrochemical test strip for the simultaneous detection of inorganic phosphorus and salicylic acid includes: An insulating substrate includes a first insulating substrate 1 and a second insulating substrate 7 disposed opposite to each other; The conductive film layer includes a first conductive film layer 2 disposed on the inner surface of the first insulating substrate 1 and a second conductive film layer 6 disposed on the inner surface of the second insulating substrate 7. The first conductive film layer 2 includes a first reactant working electrode 2-1 and a second reactant working electrode 2-2 that are mutually insulated; The second conductive film layer 6 includes a first reactant reference electrode 6-3, a second reactant reference electrode 6-4, a first pin 6-5, and a second pin 6-6 that are mutually insulated; the conductive lines of the first reactant working electrode 2-1 and the first reactant reference electrode 6-3 are staggered with the conductive lines of the second reactant working electrode 2-2 and the second reactant reference electrode 6-4. The second insulating substrate 7 has a first through hole 7-1 and a second through hole 7-2. The first working electrode 2-1 is electrically connected to the first pin 6-5 through the first through hole 7-1, and the second working electrode 2-2 is electrically connected to the second pin 6-6 through the second through hole 7-2. The reaction membrane layer 3 includes a first reaction membrane 3-1 and a second reaction membrane 3-2. The first reaction membrane 3-1 covers the first reactant working electrode 2-1, and the second reaction membrane 3-2 covers the second reactant working electrode 2-2. Reaction enzyme layers are provided on the first reaction membrane 3-1 and the second reaction membrane 3-2. Liquid guiding tank 4 is disposed between the first insulating substrate 1 and the second insulating substrate 7. Liquid guiding tank 4 includes a first liquid guiding tank 4-1 and a second liquid guiding tank 4-4 that are independent of each other. The first liquid guiding tank 4-1 is provided with a first sample inlet 4-2, and the second liquid guiding tank 4-4 is provided with a second sample inlet 4-3. The first reaction membrane 3-1 is located in the first liquid guiding tank 4-1, and the second reaction membrane 3-2 is located in the second liquid guiding tank 4-4. The hydrophilic membrane layer 5 includes a first hydrophilic membrane 5-1 and a second hydrophilic membrane 5-2, which respectively cover the first liquid guiding groove 4-1 and the second liquid guiding groove 4-4 to promote capillary flow of the sample in the first liquid guiding groove 4-1 and the second liquid guiding groove 4-4. The second insulating substrate 7 is also provided with a first sample inlet observation window 6-1 and a second sample inlet observation window 6-2. The first sample inlet observation window 6-1 corresponds to the position of the first sample inlet 4-2, and the second sample inlet observation window 6-2 corresponds to the position of the second sample inlet 4-3.

[0022] In the structure of the aforementioned electrochemical test paper, the first liquid guiding groove 4-1 and the second liquid guiding groove 4-4 are independent of each other, each with its own independent sample inlet (first sample inlet 4-2, second sample inlet 4-3), and the first reaction membrane 3-1 and the second reaction membrane 3-2 are isolated from each other and do not interfere with each other. This allows the same test paper to simultaneously detect both inorganic phosphorus and salicylic acid. Furthermore, the working electrode (first conductive film layer 2) of the electrochemical test paper of this invention is disposed on the inner surface of the first insulating substrate 1; the reference electrode and lead (second conductive film layer 6) are disposed on the inner surface of the second insulating substrate 7. This face-to-face arrangement of the working electrode and the reference electrode shortens the electrode spacing and improves the response speed and signal intensity of the electrochemical reaction.

[0023] The preparation method of the above-mentioned electrochemical test paper includes the following steps: (1) The raw materials for the reaction enzyme layer of the first reaction membrane, namely 25 KU hypoxanthine oxidase, 65 KU purine nucleoside phosphorylase, 2.5 g N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt, 3.5 g hypoxanthine nucleoside, 1.5 g sodium alginate, 5 g trehalose, 1.5 g triton X-100, and 1 g potassium ferrocyanide, were added to 1 L 0.5 M phosphate buffer with a pH of 7.2 and mixed and stirred for 1 hour to obtain the first enzyme slurry; the raw materials for the reaction enzyme layer of the second reaction membrane, namely 15 KU salicylate hydroxylase, 55 KU tyrosinase, 1.2 g 3-methyl-2-benzothiazolinone hydrazone hydrochloride, 3 g reduced nicotinamide adenine dinucleotide, 1.5 g sodium alginate, 5 g trehalose, 1.5 g triton X-100, 1 g potassium ferrocyanide, and 1 L 0.5 M phosphate buffer, were added to phosphate buffer with a pH of 7.2 and stirred for 1 hour to obtain the first enzyme slurry; The first enzyme slurry was obtained by mixing and stirring in a phosphate buffer solution with a pH of 7.5 for 1 hour; the second enzyme slurry was then fixed onto the first reaction membrane using an enzyme application machine, and the second enzyme slurry was fixed onto the second reaction membrane. (2) Print the first conductive film layer on the first insulating substrate, print the second conductive film layer on the second insulating substrate, and dry at 55°C for 25 min; (3) Fix the first reaction membrane containing enzyme slurry on the first conductive film layer; fix the second reaction membrane containing enzyme slurry on the second conductive film layer; attach the liquid guiding groove to the insulating substrate, and attach the hydrophilic film layer on the liquid guiding groove body to obtain the finished electrochemical test paper.

[0024] Example 2 The structure of the two-in-one electrochemical test paper used in this embodiment for simultaneous detection of inorganic phosphorus and salicylic acid is the same as that in Example 1, and the preparation method is as follows: (1) The raw materials for the first reaction membrane's reaction enzyme layer, namely 30 KU hypoxanthine oxidase, 65 KU purine nucleoside phosphorylase, 2.5 g N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt, 3.5 g hypoxanthine nucleoside, 1.5 g sodium alginate, 5 g trehalose, 1.5 g triton X-100, and 1 g potassium ferrocyanide, were added to 1 L of 0.5 M phosphate buffer with a pH of 7.5 and mixed and stirred for 1 hour to obtain the first enzyme slurry; the raw materials for the second reaction membrane's reaction enzyme layer, namely 20 KU salicylate hydroxylase, 55 KU tyrosinase, 1.2 g 3-methyl-2-benzothiazolinone hydrazone hydrochloride, 3 g reduced nicotinamide adenine dinucleotide, 1.5 g sodium alginate, 5 g trehalose, 1.5 g triton X-100, 1 g potassium ferrocyanide, and 1 L of 0.5 M phosphate buffer, were added to 1 L of phosphate buffer with a pH of 7.5 ... The first enzyme slurry was obtained by mixing and stirring in a phosphate buffer solution with a pH of 7.5 for 1 hour; the second enzyme slurry was then fixed onto the first reaction membrane using an enzyme application machine, and the second enzyme slurry was fixed onto the second reaction membrane. (2) Print the first conductive film layer on the first insulating substrate, print the second conductive film layer on the second insulating substrate, and dry at 50°C for 30 min; (3) Fix the first reaction membrane containing enzyme slurry on the first conductive film layer; fix the second reaction membrane containing enzyme slurry on the second conductive film layer; attach the liquid guiding groove to the insulating substrate, and attach the hydrophilic film layer on the liquid guiding groove body to obtain the finished electrochemical test paper.

[0025] Example 3 The structure of the two-in-one electrochemical test paper used in this embodiment for simultaneous detection of inorganic phosphorus and salicylic acid is the same as that in Example 1, and the preparation method is as follows: (1) The raw materials for the reaction enzyme layer of the first reaction membrane, namely 25 KU hypoxanthine oxidase, 55 KU purine nucleoside phosphorylase, 2.5 g N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt, 3.5 g hypoxanthine nucleoside, 1.5 g sodium alginate, 5 g trehalose, 1.5 g triton X-100, and 1 g potassium ferrocyanide, were added to 1 L 0.5 M phosphate buffer with a pH of 7.2 and mixed and stirred for 1 hour to obtain the first enzyme slurry; the raw materials for the reaction enzyme layer of the second reaction membrane, namely 15 KU salicylate hydroxylase, 45 KU tyrosinase, 1.2 g 3-methyl-2-benzothiazolinone hydrazone hydrochloride, 3 g reduced nicotinamide adenine dinucleotide, 1.5 g sodium alginate, 5 g trehalose, 1.5 g triton X-100, 1 g potassium ferrocyanide, and 1 L 0.5 M phosphate buffer, were added to 1 L phosphate buffer with a pH of 7.2 and stirred for 1 hour to obtain the first enzyme slurry; The first enzyme slurry was obtained by mixing and stirring in a phosphate buffer solution with a pH of 7.5 for 1 hour; the second enzyme slurry was then fixed onto the first reaction membrane using an enzyme application machine, and the second enzyme slurry was fixed onto the second reaction membrane. (2) Print the first conductive film layer on the first insulating substrate, print the second conductive film layer on the second insulating substrate, and dry at 60°C for 20 min; (3) Fix the first reaction membrane containing enzyme slurry on the first conductive film layer; fix the second reaction membrane containing enzyme slurry on the second conductive film layer; attach the liquid guiding groove to the insulating substrate, and attach the hydrophilic film layer on the liquid guiding groove body to obtain the finished electrochemical test paper.

[0026] Comparative Example 1 The structure of the two-in-one electrochemical test paper used in this example for the simultaneous detection of inorganic phosphorus and salicylic acid is the same as that in Example 1. During preparation, the raw material of the reaction enzyme layer does not contain L triton X-100, and other methods are the same as in Example 1.

[0027] Comparative Example 2 The structure of the two-in-one electrochemical test strip used in this example for the simultaneous detection of inorganic phosphorus and salicylic acid is the same as that in Example 1. During preparation, the raw material of the reaction enzyme layer does not contain reduced nicotinamide adenine dinucleotide, and other methods are the same as in Example 1.

[0028] The methods of using the test strips prepared in the above embodiments and comparative examples are as follows: (1) Insert the electrode pin at the end of the test strip into the corresponding test slot of the analyzer.

[0029] (2) Add the sample to be tested to the two inlets at the front of the test strip (i.e., the first inlet 4-2 and the second inlet 4-3). The sample volume per channel is about 2~5μL. The sample volume should be enough to completely fill the liquid guide groove and fully wet the reaction membrane layer. After the sample is added, it will be automatically guided by the liquid guide groove (the first liquid guide groove 4-1 and the second liquid guide groove 4-4) through the siphon effect and reach the respective enzyme reaction membrane layer (the first reaction membrane 3-1 is the inorganic phosphorus reaction layer and the second reaction membrane 3-2 is the salicylic acid reaction layer).

[0030] (3) After the sample is added, the instrument automatically triggers timing and detection. The electrical signal generated by the enzyme reaction is collected by the conductive layer and transmitted to the analyzer, and the quantitative result is directly output.

[0031] Result determination instructions: Inorganic phosphorus: The linear detection range is approximately 0.65–3.5 mmol / L. If the instrument output concentration falls within this range, it indicates the presence of inorganic phosphorus in the sample. The normal reference range for serum inorganic phosphorus in adults is 0.81–1.45 mmol / L. A concentration below 0.81 mmol / L indicates hypophosphatemia, and a concentration above 1.45 mmol / L indicates hyperphosphatemia.

[0032] Salicylic acid: The detection linear range is approximately 0.3–5.0 mmol / L. If the instrument output concentration falls within this range, it indicates the presence of salicylic acid in the sample. Clinically, it can be used for monitoring blood drug concentrations and screening for toxicity of salicylic acid-based drugs.

[0033] The test strips prepared in Examples 1 to 3, and Comparative Examples 1 and 2 were evaluated by randomly sampling 15 venous blood samples at varying intervals for testing, and then comparing them with hospital biochemical values. The results are shown in Tables 1-5. Table 1. Comparison of detection results of the test strip prepared in Example 1 with hospital biochemical values. The test strip prepared in Example 1 was used in conjunction with the LBM-01 multi-parameter analyzer for testing. Fifteen venous blood samples with varying gradients were randomly drawn for testing. The results were very close to the hospital biochemical values. The R-squared value for inorganic phosphorus was 0.997, and the R-squared value for salicylic acid was 0.998, indicating that the test strip results of this invention have a good correlation with the biochemical analyzer results.

[0034] Table 2. Comparison of the detection results of the test strips prepared in Example 2 with hospital biochemical values. The test strips prepared in Example 2 were used in conjunction with the LBM-01 multi-parameter analyzer for testing. Fifteen venous blood samples with varying gradients were randomly drawn for testing. The results were then compared with the hospital's biochemical values. The R-squared values ​​for inorganic phosphorus and salicylic acid were both above 0.999, indicating that the test results matched the hospital's test results well.

[0035] Table 3. Comparison of the detection results of the test strip prepared in Example 3 with hospital biochemical values. The test strips prepared in Example 3 were used in conjunction with the LBM-01 multi-parameter analyzer for testing. Fifteen venous blood samples with varying gradients were randomly drawn for testing. The results were then compared with the hospital's biochemical values. The R-squared value for inorganic phosphorus was 0.988, and the R-squared value for salicylic acid was 0.989, indicating that the test results were correlated with the hospital's test results. However, the results differed significantly from those of the test strips prepared in Example 2.

[0036] Table 4. Comparison of the test results of the test strips prepared in Comparative Example 1 with the hospital's biochemical values. The test strips prepared in Comparative Example 1 were used in conjunction with the LBM-01 multi-parameter analyzer for testing. Fifteen venous blood samples with varying gradients were randomly sampled and tested. The results were then compared with hospital biochemical values. The R-squared value for inorganic phosphorus was 0.970, and the R-squared value for salicylic acid was 0.979, indicating that Triton X-100 had a significant impact on accuracy.

[0037] Table 5. Comparison of the test results of the test strips prepared in Comparative Example 2 with the hospital's biochemical values. The test strips prepared in Comparative Example 2 were used in conjunction with the LBM-01 multi-parameter analyzer for testing. Fifteen venous blood samples with varying gradients were randomly drawn and tested. The results were then compared with hospital biochemical values. The R-squared value for inorganic phosphorus was 0.996, and the R-squared value for salicylic acid was 0.976. This indicates that reduced nicotinamide adenine dinucleotide has a significant impact on the accuracy of salicylic acid testing. The accuracy of salicylic acid detection is poor when reduced nicotinamide adenine dinucleotide is missing.

[0038] In summary, by improving the structure and reaction enzyme layer of the electrochemical test paper, this invention enables the electrochemical test paper to be used in conjunction with a multi-parameter analyzer, allowing for the simultaneous and rapid quantitative detection of the content of inorganic phosphorus and salicylic acid in whole blood, serum / plasma, and other body fluids in humans or animals. Furthermore, the detection results show good matching with the results of the biochemical analyzer, resulting in high efficiency.

[0039] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A two-in-one electrochemical test strip for simultaneous detection of inorganic phosphorus and salicylic acid, characterized in that, include: An insulating substrate includes a first insulating substrate and a second insulating substrate disposed opposite to each other; A conductive film layer, the conductive film layer comprising a first conductive film layer disposed on the inner surface of the first insulating substrate and a second conductive film layer disposed on the inner surface of the second insulating substrate; The first conductive film layer includes a first reactant working electrode and a second reactant working electrode that are insulated from each other. The second conductive film layer includes a first reactant reference electrode, a second reactant reference electrode, a first pin, and a second pin, which are mutually insulated from each other; The second insulating substrate has a first through hole and a second through hole. The first reactive working electrode is electrically connected to the first pin through the first through hole, and the second reactive working electrode is electrically connected to the second pin through the second through hole. The reaction membrane layer includes a first reaction membrane and a second reaction membrane, wherein the first reaction membrane covers the first reactant working electrode and the second reaction membrane covers the second reactant working electrode; and a reaction enzyme layer is disposed on the first reaction membrane and the second reaction membrane. A liquid guiding tank is disposed between the first insulating substrate and the second insulating substrate. The liquid guiding tank includes a first liquid guiding tank and a second liquid guiding tank that are independent of each other. The first liquid guiding tank is provided with a first sample inlet, and the second liquid guiding tank is provided with a second sample inlet. The first reaction membrane is located in the first liquid guiding tank, and the second reaction membrane is located in the second liquid guiding tank. A hydrophilic membrane layer, comprising a first hydrophilic membrane and a second hydrophilic membrane, which respectively cover the first liquid guiding groove and the second liquid guiding groove; The second insulating substrate is also provided with a first sample inlet observation window and a second sample inlet observation window. The first sample inlet observation window corresponds to the position of the first sample inlet, and the second sample inlet observation window corresponds to the position of the second sample inlet.

2. The electrochemical test paper according to claim 1, characterized in that, The enzyme layer of the first reaction membrane comprises: 25-35 KU / L hypoxanthine oxidase, 60-80 KU / L purine nucleoside phosphorylase, 1-10 g / L N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt, 1-10 g / L hypoxanthine nucleoside, 1-10 g / L film-forming agent, 1-20 g / L stabilizer, 1-10 g / L surfactant, 1-10 g / L electronic medium, and 0.1-0.5 M buffer solution.

3. The electrochemical test paper according to claim 1, characterized in that, The reaction enzyme layer of the second reaction membrane comprises: 15-20 KU / L salicylate hydroxylase, 50-100 KU / L tyrosinase, 1-10 g / L 3-methyl-2-benzothiazolinone hydrazone hydrochloride, 1-10 g / L reduced nicotinamide adenine dinucleotide, 1-10 g / L film-forming agent, 1-20 g / L stabilizer, 1-10 g / L surfactant, 1-10 g / L electronic medium, and 0.1-0.5 M buffer.

4. The electrochemical test paper according to claim 2 or 3, characterized in that, The film-forming agent is sodium alginate; the stabilizer is trehalose; and the surfactant is Triton X-100.

5. The electrochemical test paper according to claim 2 or 3, characterized in that, The electronic medium is potassium ferrocyanide; the buffer solution is phosphate buffer.

6. The electrochemical test paper according to claim 1, characterized in that, The conduction paths of the first reactant working electrode and the first reactant reference electrode are misaligned with the conduction paths of the second reactant working electrode and the second reactant reference electrode.

7. A method for preparing an electrochemical test paper as described in any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Mix and stir the raw materials of the enzyme layer of the first reaction membrane to obtain the first enzyme slurry; mix and stir the raw materials of the enzyme layer of the second reaction membrane to obtain the second enzyme slurry; use an enzyme dispensing machine to fix the first enzyme slurry on the first reaction membrane and fix the second enzyme slurry on the second reaction membrane; (2) Print a first conductive film layer on a first insulating substrate, print a second conductive film layer on a second insulating substrate, and dry; (3) Fix the first reaction membrane containing enzyme slurry on the first conductive film layer; fix the second reaction membrane containing enzyme slurry on the second conductive film layer; attach the liquid guiding groove to the insulating substrate, and attach the hydrophilic film layer on the liquid guiding groove body to obtain the finished electrochemical test paper.

8. The method for preparing an electrochemical test paper according to claim 7, characterized in that, The drying temperature is 50-60℃ and the drying time is 20-30 minutes.

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